Generally speaking, this invention relates to processes and apparatus for receiving blood samples for the subsequent testing of the components thereof. More particularly, this invention relates to processes and devices for the recovery of bacteria from blood by lysis and centrifugation.
Concentration of organisms by centrifugation has been well known for a number of years, such as Mycobacterium from sputum, and the obtaining of bacteria from spinal fluids, and so forth. In recent years, developments have been made of specific devices for receiving and processing blood samples, which devices are configured in such a way and contain certain components for the subsequent lysis and separation of the components of the blood sample by centrifugation. Representative of these recent developments are U.S. Pat. Nos. 4,164,449; 4,131,512 and 3,883,435.
These developments include using purified saponin, detoxified by either gel filtration or passing through a filter, in order to obtain a specific pore size through ultrafiltration. The procedures taught in these patents include testing for toxicity of saponin prior to its introduction into the devices for the subsequent lysis and separation procedures. Representative of other arrangements involved in these patents include the use of a liquid cushioning agent and an angled surface positioned in the separation device in order to provide appropriate separation of the various components of the sample, once centrifugation has taken place.
Difficulties may arise in the use of the arrangements as taught in these patents, including a double-ended tube for the introduction and removal of components from the tube, in that the layers are separated only by liquid cushioning agent type procedures and so forth. Thus, control of the removal of the supernatant is difficult due to the necessary manipulation, or accidental refluxing by the user, causing further dilution and potential loss of organisms prior to the actual separation of the layers in the devices. In most instances, the double-ended tube is required.
With this invention, by contrast, a device is provided for receiving blood samples, which device includes certain components in combination with physical beads or cylinders configured to prevent disturbing the sediment following centrifugation, and during withdrawal of the supernatant. Removal of the supernate using a pre-packaged arrangement, in accordance herewith, provides simple removal of the supernatant from the centrifuged sample with fewer manipulations. Also, prior detoxification and filtering of the saponin in the receiving container is not required.
In addition, the arrangement herein contains a combination of certain nutrients which facilitate the growth of organisms in the event of a delay in the processing of the sample. The sample may be maintained over a period of time between the time the sample is taken and the time when it is actually conveyed to a laboratory for testing, thus, removing the danger of the organisms to be tested having been destroyed by the contents of the container over a period of time prior to any testing thereof.
Thus, the invention herein includes a prepackaged evacuated container containing non-purified and non-detoxified saponin. This may be obtained from conventional sources such as, for example, practical grade saponin from Eastman Chemical Company or Fisher Scientific Company. While it is appropriate, as will be understood by practitioners-in-the-art, to pretest saponin prior to its introduction into the evacuated container because batches of saponin will vary, it is not necessary to purify, filter or detoxify the saponin prior to its introduction. The inventors have discovered, also, that by including certain other additives or nutrients in the device of the invention, the growth of organisms is facilitated in the event of a delay in processing the sample. Such materials, as discussed in more detail below, include, for example, yeast extract or other peptones, amino acids and co-enzymes.
Also included in the container are particles in the form of beads, pellets or cylinders which serve to enhance the separation during centrifugation of the components of the sample to be rested. Due to the solid or physical properties of the beads or other configured particles introduced into the evacuated tube for receiving the sample, there is less opportunity for the user to disturb the microbial sediment or concentrate during transfer of the specimen from the centrifuge to the work area.
The invention here includes, in addition to the pre-packaged evacuated container for receiving the blood sample, a pre-packaged supernate and sediment removal unit for use by the laboratory in obtaining the appropriate separated components of the sample once lysis and centrifugation have taken place. The pre-packaged arrangement includes a supernate transfer needle for penetration of the evacuated container containing the sample. This needle may be used once centrifugation has taken place for withdrawing the supernate from the original sample receiving container. The package also includes a vent needle which is inserted prior to the removal of the supernate in order to facilitate this removal. In addition, the package includes an evacuated sterile container for receiving the supernate. Finally, the package includes a needle and syringe for removing aliquots of the remaining concentrate in the original sample containing container for distribution on appropriate culture plates.
Thus, as purely illustrative of a procedure which may be utilized, in accordance with this invention, first blood is collected from a patient by drawing directly into an evacuated pre-packaged container The pre-packaged container contains a specific formulation for enhancing the maintenance of the sample during a period prior to the sample being received and processed in the laboratory. Subsequently, on arrival in the laboratory, the sample is centrifuged. A representative centrifuging process would be at 3000-3500XG for 30-45 minutes.
Subsequently, the top of the stopper of the centrifuged container is disinfected and the vent needle from the separate packaging, in accordance herewith, is introduced into the stopper. Then the supernate transfer needle is made to penetrate the stopper. It will be understood that the needle is of a sufficient length to be introduced into the container to the level of the beads or cylinders or other material utilized for the physical separation procedure. A second evacuated container, which is sterile, is removed from this package. This evacuated container will have sufficient vacuum to draw nine to ten milliliters from the original centrifuged container.
After the supernate is withdrawn, the supernate tube and transfer needle are removed. However, the vent needle is left in place. The tube containing the supernate, as will be understood, may be removed from the transfer unit and stored. The tube containing the beads or cylinders and microbial concentrate is then vortexed. Subsequent to this procedure, the tube is inverted and the beads are tapped to the stopper end along with the solution of concentrate. Then, utilizing the needle and syringe from the very same package, the sample is removed and aliquots of the concentrate are distributed, as mentioned above, on appropriate culture plates.
As stated above, one of the significant aspects of this invention is the introduction in the sample receiving container of a component which maintains and facilitates growth of microorganisms, when present in the sample prior to its being received and worked upon by a laboratory. Representative formulations include, for example, a yeast extract present within the range of between about 0.5-3 grams, and preferably 1 gram, sodium polyanethole sulfonate present within the range of between about 0.8-1.5 grams and preferably 1 gram, a practical grade non-purified and non-detoxified saponin present within the range of between about 10 and 15 grams, and, preferably, 10 grams, and IsoVitaleX.TM., a product of BBL Laboratories, Division of Becton Dickinson and Company, Baltimore, Maryland, present within the range of between about 1-3 milliliters, and preferably one milliliter, with the above four components dissolved in 100 milliliters of water. The IsoVitaleX.TM. is a nutrient component containing nicotinamide adenine dinucleotide, glutamine, vitamin B12 , and a sulfur containing amino acid such as cysteine. Added to the above formulation is sufficient sodium bicarbonate to adjust the pH to between 6.9 and 7.4.
As further illustrative of components which may be added to the evacuated container the formulation above may have added to it Rhozyme.RTM. proteases within the range of between about 0.4 gram, and preferably 5 gram. The Rhozyme.RTM. proteases usually selected will be Rhozyme 41.RTM. concentrate, a product of Rhome and Haas, and which is a proteolytic agent.
Representative formulations for addition to an evacuated container, containing the beads or cylinders, for subsequent lysing and centrifugation are listed below.
______________________________________ FORMULA 1 1 gram yeast extract 1 gram sodium polyanethole sulfonate 10 grams saponin-practical grade- non detoxified 1 milliliter IsoVitalex .TM. ______________________________________
The above four components are dissolved in 100 milliliters of water and approximately 1.6 grams of sodium bicarbonate is added to adjust the pH to within the range of between about 6.9 and 7.4.
______________________________________ FORMULA 2 1 gram yeast extract 1 gram sodium polyanethole sulfonate 1 milliliter IsoVitaleX .TM. 10 grams saponin 0.5 grams Rhozyme 41 .RTM. ______________________________________
The above five components are dissolved in 100 milliliters of water and sufficient sodium bicarbonate is added to adjust the pH to within the range of 6.9 to 7.4.
With respect to the beads or cylinders or other small objects introduced into the evacuated container for receiving the sample initially, the beads or cylinders may be comprised of plastic or a glass material, and they must be selected to be of a size which does not pack and restrict the sedimentation of bacteria at the bottom of the tube during centrifugation. Examples of satisfactory materials are cylinders having a length within the range of between about 3 and 5 mm. and a diameter within the range of between about 2 and 3 millimeters. Beads may have a representative dimension within the range of between 2 and 5 mm. in diameter. Representative materials include polycarbonate such as Lexan, a product of General Electric Company, or polytetrafluoroethylene.
Other objects and advantages of this invention will be apparent from the following description, the accompanying drawings and the appended claims.